Automated cytology systems such as the NeoPath Autopap 300 (TM) automated cytology system available from NeoPath Corporation of Bellevue, Wash. analyze an image of a biological specimen for evidence of objects such as squamous intraepithelial lesions and cancerous cells. The biological specimen is typically fixed to a microscope slide where the microscope slide includes a coverslip over the biological specimen. It is advantageous to detect the microscope slide coverslip prior to the analysis of the biological specimen for a number of reasons. The image of the specimen is considered of highest quality, exhibiting regular and uniform optical properties only if it is contained under the coverslip. When applied, the coverslip position is invariant with respect to the sample, and, as a result, the coverslip may be used as a fiducial optical element for locating specific objects in the sample.
In automated cytology systems, microscope objective lenses are designed to image specimens under a coverslip of consistent thickness. Thus, images acquired from any biological specimens not lying under the coverslip lack the proper optical properties necessary for high quality image analysis. Therefore, the location of the coverslip must be detected with a very high degree of certainty, or the entire slide may be rejected as unsatisfactory for analysis.
In some imaging applications the coverslip may span more than one field of view. Traditional edge detection techniques do not address the analysis of a small number of partial edges for reconstruction of a coverslip object spanning multiple fields of view.
In the prior art, locating a coverslip by its edges has proven to be difficult for several reasons. For example, coverslips are typically applied by hand, as a result, the quantity of adhesive used tends to vary from slide to slide. In some cases the adhesive does not reach the edge of the coverslip and voids are created in the adhesive. The edges of the voids may appear much like coverslip edges. In other cases, the adhesive may ooze out from under the coverslip causing dirt to collect on the adhesive which results in the optical properties around the edge changing.
In addition, the specimen may have structures that, in some cases, resemble edges. Such structures may degrade or prevent the successful location of the coverslip.
In a typical slide the coverslip and adhesive ranges in size from about 0.10 mm to 0.14 mm thick. The average coverslip with adhesive is approximately 0.12 mm thick. The slide is approximately 1.0 mm thick. The differences in thickness between the slide and the coverslip lead to the slide forming a much stronger edge image which tends to overwhelm the image formed by the coverslip edge when magnified by the microscope.
Traditional edge detection techniques also have difficulty in differentiating transition areas from slide edges. For example, microscope specimens typically have an opaque label attached at one end of a slide. The transition area between the opaque label and the transparent slide creates a very strong edge image. Traditional edge detection techniques often detect this strong edge image better than the weaker images of the edges of the coverslip.
Since the position of a coverslip may vary, it is necessary to accurately locate the coverslip on each slide before a slide specimen can be analyzed. For acceptable results, analysis must take place more than a predetermined distance away from the coverslip edges. Further still, data may only be considered valid if it is acquired from under the coverslip.
Further, since slide analysis requires that a data plane of material on the slide be measured at a number of positions, a map is usually developed to estimate correct focus positions. Because a microscope objective lens is typically corrected for spherical aberrations associated with a particular coverslip and adhesive thickness, focus positions are only meaningful if focused through the coverslip and adhesive. Therefore, focus map positions must be measured through the coverslip.
Further still, corners of the coverslip may be used as fiducial marks for defining a coordinate system to locate objects on the slide. An error even as small as one degree in calculating the angle of the edge line, from the center of a 60 mm coverslip yields an error at the coverslip extreme of 0.52 mm. Therefore, a high degree of accuracy and repeatability is necessary to define coordinates which are useful for precisely locating such objects. It is one motivation of the invention to provide an apparatus to locate a microscope slide coverslip spanning multiple fields of view to provide a longer basis for more accurately determining the coverslip position.